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Genome-wide CIITA-binding profile identifies sequence preferences that dictate function versus recruitment.

Scharer CD, Choi NM, Barwick BG, Majumder P, Lohsen S, Boss JM - Nucleic Acids Res. (2015)

Bottom Line: Nearly all CIITA-bound sites were within regions containing accessible chromatin, and CIITA's presence at these sites was associated with increased histone H3K27 acetylation, suggesting that CIITA's role at these non-regulated loci may be to poise the region for subsequent regulation.Computational genome-wide modeling of the CIITA bound XY box motifs provided constraints for sequences associated with CIITA-mediated gene regulation versus binding.These data therefore define the CIITA regulome in B cells and establish sequence specificities that predict activity for an essential regulator of the adaptive immune response.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322, USA.

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CIITA dependent and independent target genes. Total RNA from three independent isolations of Raji, RJ2.2.5, CIITA complemented RJ2.2.5 cells (RJ-CIITA) and CIITAΔex3 cells (see Figure 5) was extracted and the expression of genes with validated CIITA-binding sites analyzed by qRT-PCR. mRNA levels were normalized to 18s rRNA levels and plotted with respect to the expression in Raji cells. SEM was used to represent experimental variability from three independent experiments. Student's t test was used to calculate significant differential expression between cells. P-values are indicated.
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Figure 4: CIITA dependent and independent target genes. Total RNA from three independent isolations of Raji, RJ2.2.5, CIITA complemented RJ2.2.5 cells (RJ-CIITA) and CIITAΔex3 cells (see Figure 5) was extracted and the expression of genes with validated CIITA-binding sites analyzed by qRT-PCR. mRNA levels were normalized to 18s rRNA levels and plotted with respect to the expression in Raji cells. SEM was used to represent experimental variability from three independent experiments. Student's t test was used to calculate significant differential expression between cells. P-values are indicated.

Mentions: RNA was isolated from independent preparations of the indicated cell type using the RNeasy mini prep kit (Qiagen) and quantitative real-time RT-PCR (qRT-PCR) performed as previously described (45). Primers used in this study are listed in Supplemental Table S2. 18s rRNA was measured and used to normalize between samples. For each gene, qRT-PCR was performed at the same time for all cell lines examined, creating a single dataset for each gene. These data were then used as indicated in the generation of Figure 4, Table 1 and Supplemental Figure S1. Normalized data from three independent preparations were averaged and plotted as fold over the Raji cell samples. Student's t-tests were used to determine statistical significance between cell types and variation was represented by standard error of the mean (SEM).


Genome-wide CIITA-binding profile identifies sequence preferences that dictate function versus recruitment.

Scharer CD, Choi NM, Barwick BG, Majumder P, Lohsen S, Boss JM - Nucleic Acids Res. (2015)

CIITA dependent and independent target genes. Total RNA from three independent isolations of Raji, RJ2.2.5, CIITA complemented RJ2.2.5 cells (RJ-CIITA) and CIITAΔex3 cells (see Figure 5) was extracted and the expression of genes with validated CIITA-binding sites analyzed by qRT-PCR. mRNA levels were normalized to 18s rRNA levels and plotted with respect to the expression in Raji cells. SEM was used to represent experimental variability from three independent experiments. Student's t test was used to calculate significant differential expression between cells. P-values are indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4381081&req=5

Figure 4: CIITA dependent and independent target genes. Total RNA from three independent isolations of Raji, RJ2.2.5, CIITA complemented RJ2.2.5 cells (RJ-CIITA) and CIITAΔex3 cells (see Figure 5) was extracted and the expression of genes with validated CIITA-binding sites analyzed by qRT-PCR. mRNA levels were normalized to 18s rRNA levels and plotted with respect to the expression in Raji cells. SEM was used to represent experimental variability from three independent experiments. Student's t test was used to calculate significant differential expression between cells. P-values are indicated.
Mentions: RNA was isolated from independent preparations of the indicated cell type using the RNeasy mini prep kit (Qiagen) and quantitative real-time RT-PCR (qRT-PCR) performed as previously described (45). Primers used in this study are listed in Supplemental Table S2. 18s rRNA was measured and used to normalize between samples. For each gene, qRT-PCR was performed at the same time for all cell lines examined, creating a single dataset for each gene. These data were then used as indicated in the generation of Figure 4, Table 1 and Supplemental Figure S1. Normalized data from three independent preparations were averaged and plotted as fold over the Raji cell samples. Student's t-tests were used to determine statistical significance between cell types and variation was represented by standard error of the mean (SEM).

Bottom Line: Nearly all CIITA-bound sites were within regions containing accessible chromatin, and CIITA's presence at these sites was associated with increased histone H3K27 acetylation, suggesting that CIITA's role at these non-regulated loci may be to poise the region for subsequent regulation.Computational genome-wide modeling of the CIITA bound XY box motifs provided constraints for sequences associated with CIITA-mediated gene regulation versus binding.These data therefore define the CIITA regulome in B cells and establish sequence specificities that predict activity for an essential regulator of the adaptive immune response.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322, USA.

Show MeSH
Related in: MedlinePlus